Parasitology Research (2019) 118:2773–2779 https://doi.org/10.1007/s00436-019-06430-7 FISH PARASITOLOGY - ORIGINAL PAPER Haemogregarina daviesensis sp. nov. (Apicomplexa: Haemogregarinidae) from South American lungfish Lepidosiren paradoxa (Sarcopterygii: Lepidosirenidae) in the eastern Amazon region Pedro Hugo Esteves-Silva1 & Maria Regina Lucas da Silva2 & Lucia Helena O’Dwyer2 & Marcos Tavares-Dias3 & Lúcio André Viana1 Received: 10 February 2019 /Accepted: 15 August 2019 /Published online: 27 August 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Based on morphology and morphometry of gametocytes in blood and molecular phylogenetic analysis, we described a new species of hemoparasite from the genus Haemogregarina isolated from Lepidosiren paradoxa in the eastern Amazon region. Haemogregarina daviesensis sp. nov. is characterized by monomorphic gametocytes of varying maturity stage and their dimen- sions were 16 ± 0.12 μm(range13–18) in length and 6 ± 0.97 μm(range5–8) in width. The morphological and morphometric data were not identical with other haemogregarine species from fish. All specimens of L. paradoxa analyzed were infected by H. daviesensis sp. nov. and the parasitemia level was moderate (1–28/2000 blood erythrocytes). Two sequences were obtained from L. paradoxa, and these constituted a monophyletic sister clade to the Haemogregarina species. In addition, H. daviesensis sp. nov. detected here grouped with Haemogregarina sp. sequences isolated from chelonian Macrochelys temminckii,with99% bootstrap support. This study provides the first data on the molecular phylogeny of an intraerythrocytic haemogregarine of freshwater fish and highlights the importance of obtaining additional information on aspects of the general biology of these hemoparasites in fish populations, in order to achieve correct taxonomic classification. Keywords Haemogregarine . Lungfish . Morphology . 18S rRNA gene . Hemoparasites Introduction (Machado et al. 2010; Nelson et al. 2016). This species is primarily a carnivorous fish, feeding on arthropods and snails, Lepidosiren paradoxa Fitzinger, 1837 (lungfish) is a mono- and it inhabits hypoxic shallow swamps and lakes. Lungfish type of Lepidosirenidae, which is a family of Dipnoi fish with are obligate air-breathers, but during the dry season, they are distribution restricted to South America. In Brazil, its distri- able to burrow into the mud, undergo physiological changes, butionisonlyintheAmazonandParanáRiversystems and decrease their metabolic rates, thereby inducing aestiva- tion (Almeida-Val et al. 2010). Section Editor: Astrid Holzer Haemogregarina lepidosirenis Jepps, 1927 was described by Jepps (1927)inL. paradoxa in the Gran Chaco, in * Lúcio André Viana Paraguay. Haemogregarinidae Léger, 1911 is a family of pro- [email protected] tozoa comprising three genera: Haemogregarina Danilewsky, 1885; Cyrillia Lainson, 1981; and Desseria Siddall, 1995. 1 Departamento de Ciências Biológicas e da Saúde, Universidade The species in these genera are intraerythrocytic and have Federal do Amapá. Rodovia Juscelino Kubitscheck, KM 02, S/N, Jardim Marco Zero, Macapá, Amapá 68903-419, Brazil heteroxenous life cycles (Siddall 1995). Currently, these gen- era are regarded as differing in oocyst formation and sporozo- 2 Departamento de Parasitologia, Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Filho, Distrito de ite numbers in invertebrate hosts (vectors) and vertebrate hosts Rubião Junior, Botucatu, São Paulo 18618-970, Brazil (fish and chelonians). The vectors for haemogregarines of fish 3 Embrapa Amapá, Rodovia Juscelino Kubitscheck, Km 5, 2600, are usually isopods of the family Gnathiidae (Davies and Smit Universidade, Macapá, Amapá 68903-419, Brazil 2001) and leeches of the families Glossiphoniidae and 2774 Parasitol Res (2019) 118:2773–2779 Piscicolidae (Lainson 1981; Siddall and Desser 1993). municipality of Santana, state of Amapá, in the eastern Transmission of these intracellular parasites occurs through Amazon region of Brazil (0° 02′ 10.91″ S; 51° 09′ 39.42″ sporozoite inoculation during vector hematophagy (Davies W). Blood samples of euthanized specimens by means of 1995;Siddall1995; Davies and Johnston 2000). medullary sectioning were taken by puncturing the ventral In Brazil, the only existing records of haemogregarines in pulmonary artery using insulin syringes containing 10% fish are Desseria mugili (Carini, 1932) Siddall, 1995, in Mugil EDTA. These samples were used to prepare blood smear liza Cuvier and Valenciennes, 1836; Haemogregarina slides, which were air-dried, fixed using absolute methanol platessae Lebailly, 1905, in Paralichthys orbignyanus for 3 min, and stained with 10% Giemsa for 30 min. The blood Valenciennes, 1839; and Cyrilia lignieresi Lainson, 1981, in smear slides were examined under optical microscopy using a Synbranchus marmoratus Bloch, 1795 (Siddall 1995; Davies calibrated ocular micrometer at × 1000 magnification under et al. 2008; Eiras et al. 2012); and Cyrilia sp. in cururu sting- oil immersion for morphological and morphometric character- ray (Potamotrygon cf. histrix) and Potamotrygon wallacei, ization of the haemogregarines. The following measurements from Mariuá Archipelago, Negro River, in the Brazilian were analyzed: (1) length and width of parasites; (2) dimen- Amazon Basin (Magro et al. 2016; Oliveira et al. 2017). sions of infected and non-infected erythrocytes; and (3) di- These descriptions of intracellular parasites were made mensions of the nuclei of infected and non-infected erythro- using only the morphological aspects of the hemoparasites; cytes. The parasitemia level was estimated based on examina- however, the morphological descriptions are improper and tion of 2000 erythrocytes, in 20 replicates of 100 erythrocytes uncertain because it is difficult to differentiate (Desser per field examined, on one slide from each fish from which 1993). This is a potentially important gap in the knowledge blood samples were obtained (Godfrey Jr et al. 1987, 1990). of these parasites, because there is reason to suspect that haemogregarines could be highly diversified, especially Molecular and phylogenetic analysis Haemogregarina spp. (Úngari et al. 2018). Although a recent- ly study provided the first molecular data of Haemogregarina Genomic DNA from two samples that had been found to be bigemina from marine fish (Hayes and Smit 2019), no studies positive for Haemogregarina sp. through blood smears was using molecular tools to determine the species of isolated from 200 μL of blood, using the illustra blood Haemogregarina in freshwater fish have been conducted genomicPrep Mini Spin Kit (GE Healthcare), following the and reported. There is a need to use molecular markers to manufacturer’sinstructions. delimit the species of haemogregarines and thus distinguish DNA samples were screened for the presence of the taxa and clarify the biodiversity of this group, so as to set Haemogregarina spp., using the primers HepF300 and future taxonomic work on a firm footing. Hep900, which amplify 600 bp of the 18S rRNA gene Hence, the aim of the present study was to describe a new (Ujvarietal.2004). The PCR conditions were 94 °C for species of Haemogregarina in L. paradoxa, from the eastern 3 min, 35 cycles at 94 °C for 45 s, 56 °C for 1 min, 72 °C Amazon region of Brazil, using the features of its morphology for 1 min, and a final extension step at 72 °C for 7 min. For and molecular biology. additional sequencing and phylogenetic analysis, the two pos- itive samples in the first PCR were also amplified using the 4558/2733 primer pair (Mathew et al. 2000), which target a Materials and methods larger fragment (1120 bp) of the 18S rRNA gene. The PCR conditions of the secondary PCR consisted of a pre-PCR step Authorization at 94 °C for 3 min, followed by 40 cycles of 94 °C for 1 min, 55 °C for 2 min, an extension at 72 °C for 2 min, and a final This study was developed according to the principles adopted extension at 72 °C for 10 min. A negative control (distilled by the Brazilian College of Animal Experimentation water) and a positive control (Hepatozoon canis DNA isolated (COBEA) and with the participation of the Embrapa Amapá from naturally infected dogs) were used in each reaction. The Animal Use Committee (#004-CEUA/CPAFAP) and ICMBio PCR products were purified and sequenced using the (Chico Mendes Institute of Biodiversity; SISBIO number: BigDye™ Terminator v.3.1 cycle sequencing ready reaction 37846-1). kit (Applied Biosystems, Foster City, CA, USA) and the ABI 3500 genetic analyzer (Applied Biosystems, Foster City, CA, Site of collection, fish capture, and haemogregarine USA). survey procedures The DNA sequences obtained in this study were edited using the BioEdit software v7.2.5 (Hall 1999) and were com- In June and July 2016 and February 2017, four specimens of pared for similarity to the sequences available in GenBank® L. paradoxa (three males and one female) were caught using using BLAST. Multiple alignment, for the two sequences ob- hooks and dip nets in the Igarapé Fortaleza basin, in the tained here and 25 sequences retrieved from GenBank®, was Parasitol Res (2019) 118:2773–2779 2775 performed using the MUSCLE algorithm and the GENEIOUS A phylogenetic tree (Fig. 2), inferred from a 1020 bp frag- v.7.1.3 software. Only the sequences with at least 1000 bp ment of the 18S rRNA gene from 27 sequences, showed that were used for the multiple alignment. The best evolutionary H. daviesensis sp. nov. obtained